Application of the Barton photochemical reaction in the synthesis of 1-dethia-3-aza-1-carba-2-oxacephem: A novel agent against resistant pathogenic microorganisms

Article abstract of DOI:10.1039/b304167k

Racemic 7-(phenylacetamido)-1-dethia-3-aza-1-carba-2-oxacephem 3 was synthesized and found to possess antibacterial activity against Staphylococcus aureus PDA 209P, Escherichia coli ATCC 39188, Pseudomonas aeruginosa 1101-75 and Klebsiella pneumoniae NCTC 418 as well as the β-lactamase producing organisms E. coli A9675 and P. aeruginosa 18S-H and the methicillin-resistant organism S. aureus 95. Formation of the carbacephem 3 originated from the Barton photochemical reaction in the conversion of 8 to 10. Intramolecular cyclization of syn-oximino β-lactam 10 afforded 7-azido-2-oxa-3-azacephem 11, which was reduced and acylated to 12. Enzymatic removal of the methyl group from 12 gave the target molecule 3.

Full text of DOI:10.1039/b304167k

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Application of the Barton photochemical reaction in the synthesis
of 1-dethia-3-aza-1-carba-2-oxacephem: a novel agent against
resistant pathogenic microorganisms
a,b
a
c
c
Gholam Hossein Hakimelahi,* Pai-Chi Li, Ali A. Moosavi-Movahedi, Jamshid Chamani,
d
a
a
b
a
Ghadam Ali Khodarahmi, Tai Wei Ly, Famil Valiyev, Max K. Leong, Shahram Hakimelahi,
b
a
Kak-Shan Shia and Ito Chao*
a
Institute of Chemistry, Academia Sinica, Taipei, Taiwan, 115, R. O. C.
E-mail: ichao@chem.sinica.edu.tw; Fax: ϩ886-2-27831237; Tel: ϩ886-2-27898530
TaiGen Biotechnology, 138 Shin Ming Rd., Taipei, Taiwan 114, R. O. C.
E-mail: hosein@taigenbiotech.com.tw; Fax: ϩ886-2-27963606; Tel: ϩ886-2-27901861
Institute of Biochemistry-Biophysics, Tehran University, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy,
b
c
d
Isfahan University of Medical Sciences, Isfahan, Iran
Received 23rd April 2003, Accepted 30th May 2003
First published as an Advance Article on the web 12th June 2003
Racemic 7-(phenylacetamido)-1-dethia-3-aza-1-carba-2-oxacephem 3 was synthesized and found to possess
antibacterial activity against Staphylococcus aureus FDA 209P, Escherichia coli ATCC 39188, Pseudomonas
aeruginosa 1101–75 and Klebsiella pneumoniae NCTC 418 as well as the β-lactamase producing organisms E. coli
A9675 and P. aeruginosa 18S-H and the methicillin-resistant organism S. aureus 95. Formation of the carbacephem 3
originated from the Barton photochemical reaction in the conversion of 8 to 10. Intramolecular cyclization of
syn-oximino β-lactam 10 afforded 7-azido-2-oxa-3-azacephem 11, which was reduced and acylated to 12. Enzymatic
removal of the methyl group from 12 gave the target molecule 3.
Introduction
The first generation cephalosporins (i.e., cephalexin and
cefadroxil) have excellent activity against gram-positive bac-6
Like penicillins, cephalosporins exert a certain biological activ-
ity by acylating serine residues of transpeptidases, where the
cross-linking of peptidoglycans does not take place (Scheme
teria but variable activity against gram-negative organisms.
The second generation cephalosporins (i.e., cefaclor, cefur-
7–10
oxime axetil, and cefprozil)
remain fairly active against
1,2
1
). Bacteria become resistant to antibiotics either through
gram-positive organisms while their gram-negative spectrum is
genetic mutations or by acquiring resistant genes from other
somewhat expanded compared to the first generation cephalo-
3
bacteria. The rapid development and spread of mechanisms of
6
sporins. The third generation cephalosporins (i.e., cefixime,
bacterial resistance, however, are making virtually all β-lactam
antibiotics obsolete. The main cause of bacterial resistance to
β-lactam antibiotics is the β-lactamases, which are related in
evolutionary terms to transpeptidases. Bacteria may syn-
thesize the β-lactamase in the presence of frequently used anti-
biotics. Thus to overcome the destructive action of an inducible
β-lactamase, the structure of the β-lactam (i.e., cephalosporin)
may be altered to become insensitive to hydrolysis by the
β-lactamase (not to be a substrate for β-lactamase) while
maintaining its potency as an antibiotic (to be a substrate
7–10
cefpodoxime proxetil, and ceftibuten)
are, generally, less
3
active against gram-positive organisms than the first generation
cephalosporins (satisfactory for streptococci but inadequate for
staphylococci). They are, however, extremely active against
4
6,8
enteric gram-negative bacteria including H. influenzae. The
11
fourth generation cephalosporins such as cefepime, with a
positively charged quaternary ammonium attached to the
dihydrothiazone ring, can penetrate better through the
outer membrane of gram negative bacteria and possess a
lower affinity than the third generation cephalosporins for
certain chromosomal β-lactamases of gram-negative bacilli.
Like the third generation agents, cefepime is active against
neisseria and H. influenzae, but has greater activity against
ceftazidime-resistant Pseudomonas aeruginosa and the gram-
negative enterics (i.e., Enterobacter, indol-positive Proteus,
Citrobacter, and Serratia) possessing inducible chromosomal
β-lactamase.
5
for transpeptidase). As such, generations of penicillins and
cephalosporins were developed.
6
Another new drug which is related to, but differs from, the
12
cephalosporins is loracarbef. It is a carbacephem, which
differs from cephalosporins by the substitution of a methylene
group for the sulfur atom in the six-membered ring. Carb-
acephems have remarkable chemical stability that permits
structural manipulation that can broaden the antibacterial
spectrum of the compound and also facilitate oral absorption.
Loracarbef is extremely well absorbed and has a long half-life,
permitting twice daily dosing. The drug has been used success-
fully in patients with streptococcal pharyngitis, otitis media,
pneumonia, and cystitis. Unfortunately, it is not active against
Scheme 1 Mode of action of β-lactam antibiotics with trans-
peptidases.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 6 1 – 2 4 6 7
2461

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Ϫ1
a
2,33
Table 1 Minimum inhibitory concentrations (MIC, µg mL
)
of carbacephem (±)ꢀ3, monocyclicꢀβꢀlactam (±)ꢀ17, cephalosporin (1),
37 32
2,42
34,35
36
cephalothin (18), cefotaxime (19),
penicillin G (20), imipenem (21), clavulanic acid (22), (±)ꢀ3 ϩ 22 (1 : 1 w/w) and 19 ϩ 22 (1 : 1 w/w)
against Microorganisms
S. aureus
E. coli
P. aeruginosa
K. pneumoniae
b, c
b
b
βꢀlactam
FDA 209P
95
ATCC 39188
A9675
1101–75
18SꢀH
NCTC 418
(
(
±)ꢀ3
±)ꢀ17
1
0.01 ± 0.00
98.7 ± 3.85
0.78 ± 0.11
0.31 ± 0.04
0.07 ± 0.02
0.48 ± 0.12
0.02 ± 0.00
>128
0.35 ± 0.10
>128
>128
34.3 ± 2.03
42.8 ± 1.71
>128
14.12 ± 2.10
>128
0.08 ± 0.02
>128
0.29 ± 0.10
>128
98.6 ± 3.20
29.5 ± 1.47
58.4 ± 1.58
>128
12.1 ± 0.32
>128
0.62 ± 0.12
8.74 ± 0.08
0.45 ± 0.13
93.5 ± 3.33
128
7.48 ± 0.31
69.6 ± 2.90
128
5.30 ± 0.25
>128
0.92 ± 0.14
29.0 ± 2.50
0.71 ± 0.14
128
>128
120
> 128
>128
33.7 ±2.98
>128
1.63 ± 0.20
37.1 ± 2.30
0.03 ± 0.01
>128
>128
15.31 ± 2.11
10.61 ± 1.02
>128
0.07 ± 0.02
>128
7.35 ± 0.92
0.97 ± 0.14
0.28 ± 0.03
2.78 ± 0.41
0.33 ± 0.06
>128
1
1
2
2
2
8
9
0
1
2
3
ϩ 22
0.02 ± 0.00
0.04 ± 0.01
0.66 ± 0.17
5.10 ± 0.13
0.14 ± 0.01
0.11 ± 0.02
0.05 ± 0.00
1.52 ± 0.10
1
9 ϩ 22
a
The lowest concentrations of antibiotics needed for the prevention of visible growth of microorganisms, reported as the average values of duplicate
determinations (± standard error) and were obtained by use of an agar dilution method whereby organisms were deposited onto medicated agar
3
1 b
c
plates by the replication device of Steers et al. βꢀLactamaseꢀproducing organism. Methicillinꢀresistant organism.
S. pneumoniae and βꢀlactamase producing bacterial species
such as H. influenzae and M. catarrhalis.
Since all gramꢀnegative bacteria possess a chromosomal βꢀ
lactamase gene capable of producing an inducible chromoꢀ
somal βꢀlactamase that can mediate resistance to the old peniꢀ
cillins and cephalosporins, we designed and synthesized new
carbacephem (3) and monocyclic βꢀlactam 17 having an elecꢀ
tron sink at the 3ꢀposition (see Schemes 1 and 4).
As shown in Scheme 1, ring opening of the βꢀlactam nucleus
occurs when cephalosporins (1) react with the enzymes responꢀ
sible for the cell wall synthesis of bacteria. Consequently, a
carbanion at the Cꢀ3 position of the acyl–enzyme intermediate
13
2
will be generated. When the atom at the 3ꢀposition of
cephalosporins possesses high electronegativity, the susceptiꢀ
bility of the βꢀlactam ring towards nucleophilic attack by
transpeptidases can be enhanced. As such, cephalosporins may
exhibit superior antibacterial activity. Antibacterial activity of
these novel compounds against Staphylococcus aureus FDA
2
1
09P, Escherichia coli ATCC 39188, Pseudomonas aeruginosa
101–75, Klebsiella pneumoniae NCTC 418 as well as the
βꢀlactamase producing organisms E. coli A9675, P. aeruginosa
8SꢀH and methicillinꢀresistant organism S. aureus 95 were
1
evaluated and compared to those of cephalosporins and other
classical βꢀlactams. Results from the computational studies
suggest that the location of βꢀlactam binding to the penicillin
binding proteins (i.e., ꢀAlaꢀꢀAlaꢀpeptidase) as well as the
solvation effects may be playing roles in profound antibacterial
activity of novel 1ꢀdethiaꢀ3ꢀazaꢀ1ꢀcarbaꢀ2ꢀoxacephem 3 relative
to those of classical βꢀlactams (e.g. 1) and monocyclic βꢀlactam
Scheme
Reagents: (a) 1. transꢀPhCH᎐CHCHO, 2. N
Ϫ75 ЊC, MeOH, 2. NaBH , Ϫ25 ЊC; (c) ClNO, pyridine, Ϫ20 ЊC;
2
Synthesis of 1ꢀdethiaꢀ3ꢀazaꢀ1ꢀcarbaꢀ2ꢀoxacephem 3.
CH COCl, Et N; (b) 1. O
,
3
3
2
3
4
(
d) hν, benzene, N ; (e) DEAD–P(Ph) , THF; (f ) 1. H S, Et N,
. PhCH COCl, pyridine; (g) PLE, phosphate buffer, pH 7.0, acetone.
2
2
3
2
3
2
1
7 (see Table 1).
ester 8 in 55% yield. Compound 8 proved to be stable when
protected from light below 0 ЊC. Photolysis of nitrite ester 8 in
dry benzene under a nitrogen atmosphere using a mercury lamp
gave antiꢀoximino βꢀlactam 9 (35% yield) and synꢀoximino
Results
17–19,22a
Synthesis of 7-(phenylacetamido)-1-dethia-3-aza-1-carba-
βꢀlactam 10 (25% yield).
These two regioisomers exhibꢀ
2
-oxacephem (±)-3 (Scheme 2)
ited characteristic differences in their IR absorption frequencies
of the ester carbonyl and oxime hydroxyl functionalities. Strong
intramolecular hydrogen bonding between the ester and the
oxime hydroxyl groups in 9, through a sixꢀmembered ring,
Methyl glycinate (5) was treated with transꢀcinnamaldehyde to
produce the corresponding Schiff base, which upon reaction
with azidoacetyl chloride gave racemic βꢀlactam 6 in 75% yield.
The coupling constant (5.0 Hz) of the two hydrogen atoms on
the βꢀlactam ring indicated the cis relationship of the two subꢀ
caused a significant decrease in the absorption frequencies of
Ϫ1
the ester (C᎐O; 1738 cm ) and the hydroxyl (N–OH; 3320
᎐
Ϫ1
cm ) groups relative to the corresponding absorptions of 10, in
14,15
stituents.
Ozonolysis of the styryl group in 6, followed by
which the ester and the hydroxyl groups appeared at 1750 and
16
Ϫ1
22b
reductive workup, gave alcohol 7 in 80% yield.
3620 cm , respectively. Treatment of synꢀoximino βꢀlactam
10 with diethyl azodicarboxylate (DEAD) and triphenylꢀ
17–19
The Barton reaction,
the intramolecular exchange of NO
23
and H resulting from a photolysis reaction, requires exclusively
phosphine in THF afforded bicyclic βꢀlactam 11 in 53% yield.
20
a sixꢀmembered transition state. To meet the requirement for
the application of the Barton reaction in βꢀlactam chemistry,
we decided to synthesize nitrite ester 8. Thus, alcohol 7 was
A similar reaction with antiꢀoximino βꢀlactam 9 failed and
resulted in the destruction of the βꢀlactam ring as evidenced by
the lack of an IR absorption frequency for the βꢀlactam carbꢀ
onyl. The azide group in 11 was reduced by H S and Et N, and
21
reacted with nitrosyl chloride in pyridine to generate nitrite
2
3
2
462
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 6 1 – 2 4 6 7

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Ϫ1
a
the resultant amine was acylated with phenylacetyl chloride to
Table 2 Minimum protective concentrations (MPC, µg mL ) of
24
βꢀlactams 1, (±)ꢀ3, (±)ꢀ17 and 18–22 against bacterial βꢀlactamases
βꢀLactamase of
7
ꢀ(phenylacetamido)ꢀβꢀlactam 12 in 70% yield. Finally, we
demethylated 12 with pig liver esterase (PLE, EC 3.1.1.1) to
25
produce carbacephem 3 in 35% yield.
βꢀLactam
S. aureus 95
E. coli A9675
P. aeruginosa 18SꢀH
Mechanism of the Barton photochemical reaction (Scheme 3)
(
(
±)ꢀ3
±)ꢀ17
1
97.7 ± 2.30
>128
99.8 ± 1.74
>128
85.8 ± 2.03
>128
Absorption of light by nitrite ester 8 affords activated alkoxy
radical 13 and NO radical. The activated radical abstracts
hydrogen, through a sixꢀmembered transition state, to furnish
carbon radical 14, which is then captured by NO radical to
produce the stereoisomeric oximino βꢀlactams 9 and 10 through
intermediate 15.
1.35 ± 0.23
4.01 ± 0.30
2.72 ± 0.18
1.85 ± 0.26
30.2 ± 2.94
0.36 ± 0.01
3.47 ± 0.51
6.25 ± 0.82
1.12 ± 0.07
2.02 ± 0.39
47.65 ± 3.48
5.20 ± 0.16
0.99 ± 0.05
3.70 ± 0.63
1.08 ± 0.22
1.03 ± 0.16
35.6 ± 4.89
3.65 ± 0.14
1
19
20
2
2
8
1
2
a
The average values of duplicate determinations (± standard error)
and the ability of compounds to inhibit the hydrolysis of 3ꢀ[Eꢀ(2,4ꢀ
dinitro)styryl]ꢀ(6R,7R)ꢀ7ꢀ(2ꢀthienylacetamido)ꢀ3ꢀcephemꢀ4ꢀcarboxylic
acid by βꢀlactamases from S. aureus 95, E. coli A9675 and P. aeruginosa
1
8SꢀH. MPC values, determined by the procedure of O’Callaghan
38
et al., are the lowest concentrations of βꢀlactams needed to protect
the indicator from hydrolysis by βꢀlactamases under standard test
conditions within 40 min. The hydrolysis of indicator was evidenced
by a distinct red color.
in 3, 17 and 18 survived for ∼40, ∼60 and ∼60 min, respectively;
yet at pH 12, they were destroyed within ∼5 min.
The IR absorption frequency of the carbonyl of a βꢀlactam
can be considered as a measure of its reactivity towards
Scheme 3 Mechanism of the Barton photochemical reaction in the
synthesis of oximino βꢀlactams 9 and 10.
28,29
nucleophilic attack.
βꢀLactams 3, 17 and 18 possessed an
Ϫ1
IR absorption at 1794, 1782 and 1780 cm , respectively. Conꢀ
sequently, highly strained carbacephem 3 is more susceptible
to nucleophilic attack than monocyclic βꢀlactam 17 and
cephalothin (18).
Synthesis of methyl oximino ꢀ-lactam (±)-17 (Scheme 4)
Racemic azido βꢀlactam 9 was treated with trimethylsilyl chlorꢀ
26,27
ide and NaI in refluxing CH CN,
but only 20% yield of the
3
Biological activity
desired carboxylic acid 16 was obtained and no starting
βꢀlactam 9 was recovered. A possible path for the formation of
We carried out screening experiments for the antibacterial activꢀ
30,31
1
6 is depicted in Scheme 4. All attempts, including enzymatic
ity
of βꢀlactams (±)ꢀ3, (±)ꢀ17, a mixture of (±)ꢀ3 and clavuꢀ
3,32
hydrolysis, to gain a high yield of formation of 16 failed and
resulted in the recovery or destruction of the starting material.
Silylation of the hydroxyl groups in 16 followed by reduction of
lanic acid (22) (1 : 1 w/w) as well as the reference compounds
cephalosporin (1),
2,33
2
34,35
cephalothin (18), cefotaxime (19),
36
37
penicillin G (20), imipenem (21), clavulanic acid (22) and a
mixture of 19 and 22 (1 : 1 w/w). The experiments were perꢀ
formed in vitro against different strains of four pathogenic
microorganisms. The results are summarized in Table 1. Furꢀ
thermore, we tested the βꢀlactamase inhibitory properties of
βꢀlactams (±)ꢀ3, (±)ꢀ17 and 18–22. The results are shown in
Table 2. The doses used in the above experiments were as high
the azide group with H S and Et N afforded the correspondꢀ
2
3
ing amine. Without isolation, the amine was subsequently
treated with phenylacetyl chloride and pyridine to give the
24
38
corresponding amide 17 in 46% overall yield.
Ϫ1
as 128 µg mL .
Anticellular activity
Inhibition experiments of the proliferation of human embryꢀ
onic cells (HEL) and normal fibroblasts (Hef522) by carbꢀ
47
acephem (±)ꢀ3 and cephalothin (18) were carried out. The
toxicity of the tested compounds is expressed as the cytotoxic
concentration (µM) required to reduce normal cell growth by
5
0% (CC ). For carbacephem (±)ꢀ3, CC (HEL) = 103.0 µM
5
0
5
0
and CC50 (Hef522) = 120.0 µM; yet for cephalothin (18), CC50
(
HEL) > 150 µM and CC50 (Hef522) > 150 µM. As such, the
newly synthesized carbacephem (±)ꢀ3 was found to be someꢀ
what more toxic than cephalothin (18).
Scheme 4 Synthesis of methyl oximino βꢀlactam 17. Reagents: (a) 1.
TMSCl, NaI, CH CN, 2. H O; (b) 1. TMSCl, Et N, CH Cl , 2. H S,
3
2
3
2
2
2
Et N, 3. PhCH COCl, pyridine, CH Cl .
Discussion
3
2
2
2
We found that the carbacephem (±)ꢀ3, which possesses an
electronegative nitrogen atom at the 3ꢀposition, exhibited proꢀ
found antibacterial activity (Table 1), but low βꢀlactamase susꢀ
ceptibility (Table 2). Its behavior is very different from that of
cephalosporin 1. On the other hand, methyl oximino βꢀlactam
(±)ꢀ17, a monocyclic analog of bioactive compound (±)ꢀ3, was
not a good antibacterial agent and did not show any inhibitꢀ
ory property towards βꢀlactamases. In comparison to (±)ꢀ3,
Solubility and stability of carbacephem (±)-3, monocyclic-ꢀ-
lactam (±)-17 and cephalothin (18) in water
We found that the solubility in water was 41, 50 and 25 mg
Ϫ1
mL for carbacephem 3, methyl oximino βꢀlactam 17 and
cephalothin (18), respectively; they were stable at physiological
pH for 3, 5 and 9 days, respectively. At pH 1.0, the βꢀlactam ring
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 6 1 – 2 4 6 7
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3
9–41
imipenem 21
in Table 2. As such, 21 showed much less activity relative to
±)ꢀ3 against βꢀlactamase producing microorganisms (Table 1).
was found to be less stable to the βꢀlactamases
Table 3 Solution MD results of ꢀAlaꢀꢀAlaꢀpeptidase complex with
bicyclic βꢀlactams 1 and 3
(
Difference in binding
In contrast to carbacephem (±)ꢀ3 and imipenem 21, βꢀlactams 1
and 18–20 are highly susceptible to βꢀlactamases from S. aureus
Ϫ1
energy/kJ mol
9
5, E. coli A9675, and P. aeruginosa 18SꢀH (Table 2). As a
1
3
result, they either showed minute or no activity against the
corresponding microorganisms (see Table 1). Although carbꢀ
acephem (±)ꢀ3 exhibited slightly more cellular toxicity than
cephalothin 18, it possesses much superior antimicrobial activꢀ
ity relative to 18. Clavulanic acid 22 is a highly potent inhibitor
of βꢀlactamases (Table 2), but shows no activity against microꢀ
organisms (Table 1). Therefore, it is used to examine the synꢀ
ergistic effect of binary βꢀlactams (3 ϩ 22 and 19 ϩ 22). Unlike
cefotaxime 19, carbacephem (±)ꢀ3 was not an effective subꢀ
strate for βꢀlactamases of different bacterial species (Table 2).
Consequently, the βꢀlactamase inhibitory property of clavuꢀ
lanic acid 22 did not exert any synergistic effect on (±)ꢀ3; yet it
exerted a large synergistic effect on cefotaxime 19 (see Table 1).
Although 3 and 17 were designed with the concept of generꢀ
ating an electron sink at the 3 position, density functional calcuꢀ
lation results of βꢀlactams 1, 3 and 17, however, showed that the
C᎐O NPA (natural population analysis) charges are rather simiꢀ
lar (0.715, 0.713 and 0.718 for the carbon and Ϫ0.564, Ϫ0.566
and Ϫ0.561 for the oxygen of the carbonyl groups in βꢀlactams 1,
<H> 300 K
Av stretch
Av bend
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Ϫ207.47
Ϫ0.61
Ϫ1.13
5.58
9.00
21.96
Ϫ242.28
Av torsion
Av van der Waals
Av electrostatic
Av solvation
3
and 17, respectively). It remains to be explored how the nitroꢀ
gen at the 3 position and its neighboring oxygen affect the whole
reaction path. Computational structural analysis has shown
that the lowꢀenergy conformation of 17 is very different from
those of 1 and 3. Unlike the relatively extended forms of bicyclic
βꢀlactams 1 and 3, monocyclic βꢀlactam 17 folds up due to
two pairs of intramolecular hydrogen bonds (O–H ؒ ؒ ؒ O,
N–H ؒ ؒ ؒ O; see Fig. 1). Consequently, monocyclic βꢀlactam 17
4
2a
binds to ꢀAlaꢀꢀAlaꢀpeptidase
in a position different from
those of bicyclic βꢀlactams 1 and 3. As shown in Fig. 2, the
O ؒ ؒ ؒ H distance between the carbonyl of the βꢀlactam ring in 17
and peptidase reaction site, Serꢀ62, is 4.958 Å. The corresponding
distances in peptidase–1 and peptidase–3 are 2.310 and 2.260 Å
(Fig. 3), respectively. While hydrogen bond networks between the
enzyme and substrates 1 and 3 are similar (Tyrꢀ159, Argꢀ285,
Thrꢀ299, Serꢀ62 and Thrꢀ301 residues are involved in both peptiꢀ
dase–1 and peptidase–3 complexes), the hydrogen bond network
of peptidase–17 (Fig. 2) was found to be very different from those
of peptidase–1 and peptidase–3 complexes (Fig. 3). It should be
noted that the unfolded structure of 17 had also been used in MD
simulations, but the binding affinity was found to be inferior to
the folded one shown in Fig. 2. As such, monocyclic βꢀlactam
Fig.
2
MD averaged geometry of peptidase–17 complex. The
structure of monocyclic βꢀlactam 17 is shown in black. The
distance between the hydroxyl group of Serꢀ62 and the carbonyl group
of the βꢀlactam ring is 4.958 Å.
1
7 did not exhibit activity against pathogenic microorganisms
(see Table 1) presumably due to its failure to approach the active
site in the correct orientation. While the shorter MD averaged
distances between Serꢀ62 and the βꢀlactam carbonyl groups
in bicyclic βꢀlactams 1 and 3 (ca. 2.3 Å) correlated with their
Fig. 3 MD averaged geometry of peptidase–3 complex. The structure
of carbacephem 3 is shown in black. The distance between the hydroxyl
group of Serꢀ62 and the carbonyl group of the βꢀlactam ring is 2.260 Å.
biological activity, the greater binding affinity of carbacephem 3
relative to cephalosporin 1 (Table 3) correlated with the superior
antibacterial property of 3. Interestingly, as shown in Table 3
the stronger binding affinity of carbacephem 3 was the result of
the solvation effect, rather than the nonꢀbonded interactions.
Therefore, current preliminary computational exploration of
protein–substrate binding indicates that the structure of the drug,
Fig. 1 Top view and side view of monocyclic βꢀlactam 17 calculated at
the B3LYP/6ꢀ31G* level. The OH ؒ ؒ ؒ O and NH ؒ ؒ ؒ O hydrogen
bonds are shown in the top view (left) and the side view (right),
respectively. The numbers show the H ؒ ؒ ؒ O distances as well as the
NH ؒ ؒ ؒ O and OH ؒ ؒ ؒ O angles.
2
464
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 6 1 – 2 4 6 7

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4
5
location of the drug binding, and solvation effects could be
playing roles in biological activities. A theoretical study on the
reaction path is needed to fully elucidate the electronic effect of 3
relative to 1.
mized with the GB/SA solvation model for water and then
their βꢀlactam rings were superimposed to the corresponding
part of cephalothin observed in the active site of ꢀAlaꢀꢀAlaꢀ
42a
peptidase. Before full geometry optimization of the whole
peptidase–substrate complex, the substrate was relaxed in two
ways. 1. The substrate was optimized in the peptidase structure
fixed in the Xꢀray structure of ꢀAlaꢀꢀAlaꢀpeptidase–
cephalothin. 2. The substrate was partially optimized with the
βꢀlactam ring fixed, and then fully optimized in the fixed Xꢀray
peptidase structure. Fully optimized structures of the peptiꢀ
dase–substrate complex obtained by both procedures were used
as the initial structures of MD simulations and the ones
afforded lower averaged energies are shown in Table 3 and
Figs. 2 and 3. In the MD simulations, the SHAKE algorithm
Conclusions
The application of the Barton photochemical reaction led to
the preparation of a new carbacephem antibiotic, (±)ꢀ3, with
high stability toward βꢀlactamases of different bacterial species.
Methyl oximino βꢀlactam (±)ꢀ17 was also synthesized. This
monocyclic βꢀlactam (±)ꢀ17 did not exhibit significant bioꢀ
logical activity. Results from the biological tests as well as the
computational studies indicate that the newly synthesized carbꢀ
acephem (±)ꢀ3 is an excellent substrate for transpeptidases. On
the other hand, the reference βꢀlactams 1 and 18–20 can be
recognized by both transpeptidases and βꢀlactamases. Thus, in
comparison to (±)ꢀ3, they showed no significant activity against
βꢀlactamase producing microorganisms. Moreover, imipenem
4
6
was used for all bonds to hydrogen atoms in the substrate and
the peptidase structure was fixed. The temperature was chosen
to be 300 K and the time step was 1.5 fs. The equilibration
time was 200 ps, followed by 1000 ps of averaging period. The
coordinates of structures were sampled every 2 ps during the
MD simulations.
2
1 with moderate substrate activity toward βꢀlactamases exhibꢀ
ited less activity than (±)ꢀ3 against S. aureus FDA 209P, E. coli
ATCC 39188, P. aeruginosa 1101–75 Klebsiella pneumoniae
NCTC 418 as well as the βꢀlactamase producing organisms
E. coli A9675, P. aeruginosa 18SꢀH and methicillinꢀresistant
organism S. aureus 95.
Methyl (3SR,4RS )-2-(3-azido-2-oxo-4-styryl-1-azetidinyl)-
acetate (6)
transꢀCinnamaldehyde (2.66 g, 0.0201 mol) in CH Cl (6.0 mL)
2
2
was added dropwise to a solution of methyl glycinate (5, 1.79 g,
0
.0201 mol) in CH Cl (45 mL) containing anhydrous MgSO
2
2
4
Experimental
(
3.0 g). The solution was stirred at 0 ЊC for 3.0 h, then was
filtered through Celite. Evaporation afforded a quantitative
yield of the corresponding Schiff base, which was dissolved in
CH Cl (85 mL) and Et N (5.02 g, 0.0496 mol) was added. A
General
For anhydrous reactions, glassware was dried overnight in
an oven at 120 ЊC and cooled in a desiccator over anhydrous
CaSO4 or silica gel. Reagents were purchased from Fluka
Chemical Co. Solvents, including dry ether and tetrahydrofuran
2
2
3
solution of azidoacetyl chloride (2.40 g, 0.0201 mol) in CH Cl2
2
(9.0 mL) was added dropwise over 10 min at 25 ЊC. After 2.0 h
stirring, the solution was washed with H O (2 × 80 mL), dried
2
(
THF), were obtained by distillation from the sodium ketyl
over MgSO (s), filtered, and concentrated under reduced presꢀ
4
of benzophenone under nitrogen. Other solvents, including
chloroform, dichloromethane, ethyl acetate, and hexanes were
distilled over CaH under nitrogen. Absolute methanol and
ethanol were purchased from Merck and used as received. Meltꢀ
ing points were obtained with a Büchi 510 melting point apparꢀ
atus. Infrared (IR) spectra were recorded on a PerkinꢀElmer
Paragon 1000 FourierꢀTransform spectrophotometer. The wave
sure. Purification of the residue by use of column chromatoꢀ
graphy (CH Cl as eluent) gave (±)ꢀ6 (4.32 g, 0.0151 mol) as a
2
2
2
foam in 75% yield (Found: C, 58.71; H, 4.90; N, 19.59.
1
C H N O requires C, 58.73; H, 4.93; N, 19.57%); H NMR
14
14
4
3
(CDCl ) δ 3.54 (s, 3H, CH ), 4.31 (AB q, J = 17.5, 2H, CH ),
3
3
2
4.48 (dd, J = 8.0, 5.0, 1H, HC(4)), 4.81 (d, J = 5.0, 1H, HC(3)),
6.32 (dd, J = 16.1, 8.0, 1H, CH᎐C), 6.61 (d, J = 16.1, 1H,
᎐
Ϫ1
numbers reported are referenced to the 1601 cm absorption
CHPh), 7.12–7.46 (m, 5H, C H ); IR (CH Cl ) 2100 (N ), 1765
6
5
2
2
3
Ϫ1
ϩ
of polystyrene. Proton NMR spectra were obtained on a Varian
XLꢀ300 (300 MHz) spectrometer. Chloroformꢀd and dimethyl
sulfoxideꢀd were used as solvent; Me Si (δ 0.00 ppm) was used
as an internal standard. All NMR chemical shifts are reported
as δ values in parts per million (ppm) and coupling constants
(βꢀlactam), 1745 (ester) cm ; CI–MS: 287 (M ϩ 1).
6
4
Methyl (3SR,4RS )-2-(3-azido-2-oxo-4-hydroxymethyl-1-
azetidinyl)acetate (7)
Ozone was bubbled through a solution of (±)ꢀ6 (2.16 g,
(
J) are given in hertz (Hz). The splitting pattern abbreviations
7
.55 mmol) in MeOH (75 mL) at Ϫ75 ЊC until KI–starch paper
are as follows: s, singlet; d, doublet; t, triplet; q, quartet; br,
broad; m, unresolved multiplet due to the field strength of
the instrument; and dd, doublet of doublets. Mass spectra
were carried out on a VG 70–250 S mass spectrometer. Microꢀ
analyses were performed on a Perkin–Elmer 240ꢀB microꢀ
analyzer. Purification on silica gel refers to gravity column
chromatography on Merck Silica Gel 60 (particle size 230–
showed excess O3 (∼1 h). The excess O3 was removed by
a stream of nitrogen, NaBH (1.33 g, 35.0 mmol) added at
4
Ϫ20 ЊC, and the solution allowed to warm up to 25 ЊC within
1
.0 h. Then 5% aq. HCl solution (10 mL) was added, the mixꢀ
ture concentrated, H O (60 mL) added, and extracted with
2
EtOAc (100 mL). The organic layer was then dried over MgSO4
(
s), filtered, and concentrated under reduced pressure. The
crude product was purified by use of column chromatography
CHCl as eluant) to give (±)ꢀ7 (1.29 g, 6.03 mmol) as a foam in
4
00 mesh). Analytical TLC was performed on precoated plates
purchased from Merck (Silica Gel 60 F254). Compounds were
visualized by use of UV light, I vapor or 2.5% phosphomolybꢀ
dic acid in ethanol with heating.
(
3
2
8
0% yield (Found: C, 39.14; H, 4.80; N, 26.08. C H N O
7 10 4 4
1
requires C, 39.25; H, 4.71; N, 26.16%); H NMR (CDCl –D O)
3
2
δ 3.25–3.41 (m, 2H, CH O), 3.55 (s, 3H, CH ), 3.70–4.20 (m,
Computational details
2
3
1
H, HC(4)), 4.38 (AB q, J = 18, 2H, CH ), 4.60 (d, J = 4.8, 1H,
2
Molecular mechanics (MM) and molecular dynamic (MD)
simulations were carried out with the AMBER* force field
using software MacroModel 6.5. Lowest energy structures of
the carboxylate anions of compounds 1, 3 and 17 were found by
AMBER* and used in geometry optimization at the B3LYP/
HC(3)); IR (CH Cl ) 3160 (OH), 2100 (N ), 1756 (βꢀlactam),
2
2
3
Ϫ1
ϩ
1
740 (ester) cm ; CI–MS: 215 (M ϩ 1).
43
Methyl (3SR,4RS )-2-(3-azido-2-oxo-4-nitrosooxymethyl-1-
azetidinyl)acetate (8)
44
6
ꢀ31G* level to obtain NPA charges. To prepare the peptiꢀ
dase–substrate complexes for solution phase MM and MD
calculations, the gas phase structures of 1, 3 and 17 were optiꢀ
A solution of (±)ꢀ7 (2.50 g, 11.7 mmol) in dry pyridine (35 mL)
was kept in a cooling bath at Ϫ20 ЊC and stirred, while nitrosyl
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 6 1 – 2 4 6 7
2465

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chloride was allowed to distill into the reaction flask until a
yellow color persisted in the solution (∼20 min). The solution
was partitioned between Et O (100 mL) and H O (100 mL).
eluant) gave (±)ꢀ12 (0.422 g, 1.33 mmol) as a foam in 70% yield
(Found: C, 56.71; H, 4.69; N, 13.14. C H N O requires C,
1
5
15
3
5
1
56.78; H, 4.76; N, 13.24%); H NMR (CDCl ) δ 3.56–4.21 (m,
2
2
3
The organic layer was washed with H O (4 × 100 mL), 5% aq.
2H, CH O), 3.60 (s, 2H, CH CO), 3.98 (s, 3H, CH ), 4.51–4.87
2
2
2
3
HCl solution (2 × 70 mL), dried over MgSO (s), filtered, and
(m, 1H, HC(6)), 5.03 (dd, J = 8.0, 5.0, 1H, HC(7)), 6.28 (d,
4
concentrated under reduced pressure. The crude product was
J = 8.0, 1H, NH), 7.31 (s, 5H, C H ); IR (CH Cl ) 3410 (NH),
6
5
2
2
Ϫ1
purified by use of column chromatography (CHCl as eluant) to
1798 (βꢀlactam), 1750 (ester), 1685 (amide), 1600 (C᎐N) cm ;
3
ϩ
give (±)ꢀ8 (1.56 g, 6.42 mmol) as a foam in 55% yield (Found: C,
CI–MS: 318 (M ϩ 1).
3
4.35; H, 3.60; N, 28.99. C H N O requires C, 34.57; H, 3.73;
7 9 5 5
1
N, 28.80%); H NMR (CDCl ) δ 3.61 (s, 3H, CH ), 3.62–3.70
(6RS,7SR)-8-Oxo-7-(phenylacetamido)-1,3-diaza-4-oxa-
bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (3)
3
3
(
br, 2H, CH O), 3.74–4.20 (m, 1H, HC(4)), 4.36 (AB q, J = 17.7,
2
2
1
2
H, CH ), 4.70 (d, J = 5.0, 1H, HC(3)); IR (CH Cl ) 2100 (N ),
2 2 2 3
Ϫ1
To a solution of (±)ꢀ12 (0.243 g, 0.766 mmol) in acetone
772 (βꢀlactam), 1750 (ester), 1652, 1615 (nitrite) cm ; CI–MS:
(
6.0 mL) was added 0.1 M phosphate buffer solution (pH 7.0,
ϩ
14 (M ϩ 1 Ϫ NO).
6
2
.0 mL) containing PLE (30 mg). After stirring for 12 h at
5 ЊC, the acetone was evaporated and the aq. solution was kept
Methyl (3SR,4RS )-2-(3-azido-2-oxo-4-hydroxymethyl-1-aze-
tidinyl)-2-anti-hydroxyiminoacetate (9) and the respective
syn-hydroxyiminoacetate (10)
at 10 ЊC to afford (±)ꢀ3 (0.082 g, 0.27 mmol) as pale yellow
crystals in 35% yield (Found: C, 55.32; H, 4.28; N, 13.98.
C H N O requires C, 55.44; H, 4.32; N, 13.85%); mp 132–133
14
13
3
5
1
ЊC; H NMR (CDCl –DMSOꢀd –D O) δ 3.60–4.12 (br m, 2H,
A solution of (±)ꢀ8 (2.43 g, 9.99 mmol) in benzene (170 mL)
was irradiated by a 450 W medium pressure Hanovia mercury
lamp placed inside an ice–water cooled Pyrex immersion well.
Nitrogen flow was maintained during photolysis and the
reaction temperature was kept near 10 ЊC by regulating the
temperature of the cooling water. After 1.0 h, the solvent was
concentrated under reduced pressure and the residue was puriꢀ
fied by use of column chromatography (EtOAc as eluant) to
give (±)ꢀ9 (0.849 g, 3.49 mmol, 35% yield) and (±)ꢀ10 (0.600 g,
3
6
2
CH O), 3.58 (s, 2H, CH CO), 4.45–4.83 (m, 1H, HC(6)), 5.11
2
2
(
d, J = 4.9, 1H, HC(7)), 7.28 (s, 5H, C H ); IR (CH Cl ) 3140–
6 5 2 2
3
1
665 (CO H, NH), 1794 (βꢀlactam), 1715 (C᎐O), 1680 (amide),
2
Ϫ1 ϩ
608 (C᎐N) cm ; CI–MS: 304 (M ϩ 1).
᎐
(
3SR,4RS )-2-(3-Azido-2-oxo-4-hydroxymethyl-1-azetidinyl)-2-
anti-methoxyiminoacetic acid (16)
To a solution of (±)ꢀ9 (0.485 g, 1.99 mmol) in dry CH CN
3
2
.46 mmol, 25% yield), respectively. For (±)ꢀ9 (Found: C, 34.33;
(
20 mL) was added anhydrous NaI (0.62 g, 4.0 mmol) and triꢀ
H, 3.71; N, 28.67. C H N O requires C, 34.57; H, 3.73; N,
2
7
9
5
1
5
methylsilyl chloride (0.436 g, 4.00 mmol). The mixture was
8.80%); mp 111–113 ЊC; H NMR (CDCl –D O) δ 3.31–3.56
3 2
heated at reflux temperature for 1.5 h, then H O (25 mL) was
2
(
br, 2H, CH O), 3.89 (s, 3H, CH ), 3.76–4.22 (m, 1H, HC(4)),
2
3
added and the solution was extracted with EtOAc (40 mL). The
organic layer was concentrated under reduced pressure and
the residue was purified by use of column chromatography
4
2
.65 (d, J = 5.0, 1H, HC(3)); IR (CH Cl ) 3200–3320 (OH),
2 2
Ϫ1
100 (N ), 1788 (βꢀlactam), 1738 (ester), 1640 (C᎐N) cm ; CI–
3
ϩ
MS: 244 (M ϩ 1). For (±)ꢀ10 (Found: C, 34.51; H, 3.72; N,
2
1
CH O), 3.75 (s, 3H, CH ), 3.72–4.20 (m, 1H, HC(4)), 4.60 (d,
J = 4.9, 1H, HC(3)); IR (CH Cl ) 3620, 3230 (OH), 2100 (N ),
(
EtOAc–MeOH (9 : 1) as eluant) to give (±)ꢀ16 (0.0970 g, 0.399
8.77. C H N O requires C, 34.57; H, 3.73; N, 28.80%); mp
7 9 5 5
1
mmol) in 20% yield (Found: C, 34.38; H, 3.60; N, 28.79.
17–119 ЊC; H NMR (CDCl –D O) δ 3.38–3.61 (br m, 2H,
3
2
C H N O requires C, 34.57; H, 3.73; N, 28.80%); mp 125–127
7
9
1
5
5
2
3
ЊC; H NMR (CDCl –DMSOꢀd –D O) δ 3.29–3.60 (br, 2H,
3
6
2
2
2
3
CH O), 4.01 (s, 3H, CH ), 3.81–4.28 (m, 1H, HC(4)), 4.61 (d,
2
3
Ϫ1
1
(
790 (βꢀlactam), 1750 (ester), 1605 (C᎐N) cm ; CI–MS: 244
᎐
ϩ
J = 5.0, 1H, HC(3)); IR (KBr) 3150–3400 (CO H, OH), 2100
2
M ϩ 1).
Ϫ1
(
2
N ), 1786 (βꢀlactam), 1705 (C᎐O), 1620 (C᎐N) cm ; CI–MS:
3
ϩ
44 (M ϩ 1).
Methyl(6RS,7SR)-7-azido-8-oxo-1,3-diaza-4-oxabicyclo[4.2.0]-
oct-2-ene-2-carboxylate (11)
(
3SR,4RS )-2-(2-Oxo-3-(phenylacetamido)-4-hydroxymethyl-1-
azetidinyl)-2-anti-methoxyiminoacetic acid (17)
To a solution of 10 (0.250 g, 1.03 mmol) and (Ph) P (0.270 g,
3
1
.03 mmol) in THF (8.0 mL) was added diethyl azodicarbꢀ
To a solution of (±)ꢀ16 (0.194 g, 0.798 mmol) in CH Cl2
2
oxylate (0.210 g, 1.20 mmol). The reaction mixture became red
and the color disappeared after 4 min. The mixture was stirred
at 25 ЊC for 24 h and then the solvent was concentrated under
reduced pressure. Purification of the residue by use of column
(
25 mL) and Et N (0.638 g, 6.29 mmol) was added trimethylsilyl
3
chloride (0.433 g, 3.99 mmol). The mixture was stirred at 0 ЊC
for 1.0 h. Then H S was bubbled for 5.0 min into the solution.
After stirring at 0 ЊC for 1.0 h, nitrogen was bubbled for 20 min.
Pyridine (0.128 g, 1.60 mmol) was added at 25 ЊC followed by
phenylacetyl chloride (0.247 g, 1.60 mmol) dropwise over
5.0 min. After stirring for 2.0 h, the solution was washed with
2
chromatography (CH Cl as eluant) afforded (±)ꢀ11 (0.124 g,
2
2
0
.550 mmol) as a foam in 53% yield (Found: C, 37.30; H, 3.21;
1
N, 31.13. C H N O requires C, 37.34; H, 3.13; N, 31.10%); H
7
7
5
4
NMR (CDCl ) δ 3.67–4.28 (m, 2H, CH O), 3.99 (s, 3H, CH ),
3
2
3
water (2 × 25 mL), dried over MgSO (s), filtered, and concenꢀ
4
4
.28–4.66 (m, 1H, HC(6)), 4.68 (d, J = 5.0, 1H, HC(7)); IR
trated under reduced pressure. Purification of the residue by use
of flash chromatography (EtOAc–MeOH (8.5 : 1.5)) afforded
(±)ꢀ17 (0.123 g, 0.367 mmol) in 46% yield (Found: C, 53.59; H,
(
CH Cl ) 2110 (N ), 1800 (βꢀlactam), 1754 (ester), 1598 (C᎐N)
2
Ϫ1
2
3
ϩ
cm ; CI–MS: 198 (M ϩ 1 Ϫ N ).
2
4
.97; N, 12.68. C H N O requires C, 53.73; H, 5.11; N,
15 17 3 6
1
Methyl (6RS,7SR)-8-oxo-7-(phenylacetamido)-1,3-diaza-4-oxa-
bicyclo[4.2.0]oct-2-ene-2-carboxylate (12)
12.53%); mp 131–133 ЊC; H NMR (CDCl –DMSOꢀd –D O)
3 6 2
δ 3.31–3.75 (m, 2H, CH O), 3.58 (s, 2H, CH CO), 3.99 (s, 3H,
2
2
CH ), 4.12–4.40 (m, 1H, HC(4)), 5.19 (d, J = 4.9, 1H, HC(3)),
3
To a solution of (±)ꢀ11 (0.428 g, 1.90 mmol) in CH Cl (50 mL)
2
2
7
1
.26 (s, 5H, C H ); IR (KBr) 3160–3420 (NH, CO H, OH),
6 5 2
Ϫ1
and Et N (0.193 g, 1.90 mmol) at Ϫ0 ЊC, H S was bubbled
3
2
782 (βꢀlactam), 1710 (C᎐O), 1680 (amide), 1617 (C᎐N) cm ;
᎐
᎐
for 10 min. After stirring for 1.0 h, nitrogen was bubbled for
0 min. Pyridine (0.20 g, 2.5 mmol) was added at 25 ЊC followed
by phenylacetyl chloride (0.386 g, 2.50 mmol) dropwise over
.0 min. After stirring for 1.5 h, the solution was washed with
water (3 × 40 mL) and brine (50 mL), then treated with MgSO4
s) and charcoal. Filtration, evaporation, and purification of
ϩ
CI–MS: 336 (M ϩ 1).
3
Antibacterial activity tests
The serial broth dilution method was used to study the antiꢀ
5
30
(
bacterial activity of βꢀlactams (Table 1). The inocula were
the residue by use of column chromatography (CHCl3 as
prepared by use of the heart infusion broth (Difco Laboratorꢀ
2
466
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 4 6 1 – 2 4 6 7

View Article Online
Ϫ4
ies) to make 10 dilutions of the overnight cultures. Tubes of
22 (a) R. D. Rieke and N. A. Moore, J. Org. Chem., 1972, 37, 413–418;
b) R. M. Silverstein, G. C. Bassler and T. C. Morrill in
(
the seeded antibioticꢀcontaining media were incubated at 37 ЊC
for 20 h. The lowest concentration of the βꢀlactam that preꢀ
vented visible growth of microorganisms was then determined.
Spectrometric Identification of Organic Compounds, John Wiley &
Sons, California, 1981, pp. 95–180.
2
2
3 E. Grochowski and J. Jurczak, Synthesis, 1976, 682–684.
4 G. H. Hakimelahi and G. Just, Can. J. Chem., 1981, 59, 941–944.
Toxicity test procedure in vitro
25 S. Gehanne, M. Giammaruco, M. Taddei and P. Ulivi, Tetrahedron
Lett., 1994, 35, 2047–2048.
6 T. Morita, Y. Okamoto and H. Sakurai, Tetrahedron Lett., 1978,
2523–2526.
Human embryonic cell (HEL) and normal fibroblasts (Hef522)
cell lines were cultured in Dulbecco’s modified eagle medium
2
(
DMEM) supplemented with 10% fetal bovine serum (FBS),
27 G. H. Hakimelahi and G. Just, Helv. Chim. Acta, 1982, 65, 1359–
Ϫ1
Ϫ1
1367.
2
.0 mM glutamine, 100 U mL penicillin and 100 µg mL
2
8 R. B. Morin, B. G. Jackson, R. A. Mueller, E. R. Lavagnino,
streptomycine in a humidified atmosphere with 5% CO and
2
W. B. Scanlon and S. L. Andrews, J. Am. Chem. Soc., 1969, 91, 1401–
47
2
0% O at 37 ЊC. After 24 h, carbacephem (±)ꢀ3 and cephaꢀ
2
1
407.
lothin (18), at various concentrations, were added. The cell
numbers of the control cultures as well as those cultures supꢀ
plemented with the test compounds were determined after 12,
2
9 L. J. Bellamy, The Infrared Spectra of Complex Molecules, 2nd edn.,
Wiley, New York, 1958.
30 T. A. Pursiano, M. Misiek, F. Leitner and K. E. Price, Antimicrob.
Agents Chemother., 1973, 3, 33–39.
2
4, 48, 72 and 90 h of growth. The CC50 values were estimated
3
3
3
1 F. Steers, F. L. Foltz and B. S. Graves, Antibiot. Chemother., 1959, 9,
from dose–response curves compiled from two independent
experiments and represent cytotoxic concentration (µM)
required to reduce cell growth by 50% after 90 h incubation.
3
07–311.
2 J. R. Hwu, S. Hakimelahi, A. A. MoosaviꢀMovahedi and S.ꢀC. Tsay,
Chem. Eur. J., 1999, 5, 2705–2711.
3 J. R. Hwu, A. A. Moshfegh, S.ꢀC. Tsay, C. C. Lin, W. N. Tseng,
A. Azaripour, H. Mottaghian and G. H. Hakimelahi, J. Med.
Chem., 1997, 40, 3434–3441.
4 F. J. Muhtadi and M. M. A. Hassan, in Analytical Profiles of Drug
Substances, ed. K. Florey, Academic, New York, 1982, Vol. 11,
pp. 139–168.
Acknowledgements
3
For financial support, we thank the Ministry of Science and
Higher Education of Iran, Academia Sinica, Tehran University,
and the National Science Council of Republic of China.
35 R. Wise, T. Rollason, M. Logan, J. M. Andrews and K. A. Bedford,
Antimicrob. Agents Chemother., 1978, 14, 807–811.
3
3
6 J. J. Morris and M. I. Page, J. Chem. Soc., Perkin Trans. 2, 1980,
2
12–219.
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